Condensed Matter Systems Exposed to Radiation: Multiscale Theory, Simulations, and Experiment.

This roadmap reviews the new, highly interdisciplinary research field studying the behavior of condensed matter systems exposed to radiation. The Review highlights several recent advances in the field and provides a roadmap for the development of the field over the next decade. Condensed matter systems exposed to radiation can be inorganic, organic, or biological, finite or infinite, composed of different molecular species or materials, exist in different phases, and operate under different thermodynamic conditions. Many of the key phenomena related to the behavior of irradiated systems are very similar and can be understood based on the same fundamental theoretical principles and computational approaches. The multiscale nature of such phenomena requires the quantitative description of the radiation-induced effects occurring at different spatial and temporal scales, ranging from the atomic to the macroscopic, and the interlinks between such descriptions. The multiscale nature of the effects and the similarity of their manifestation in systems of different origins necessarily bring together different disciplines, such as physics, chemistry, biology, materials science, nanoscience, and biomedical research, demonstrating the numerous interlinks and commonalities between them. This research field is highly relevant to many novel and emerging technologies and medical applications.

Spatial-Temporal Genome Regulation in Stress-Response and Cell-Fate Change.

Complex functioning of the genome in the cell nucleus is controlled at different levels: (a) the DNA base sequence containing all relevant inherited information; (b) epigenetic pathways consisting of protein interactions and feedback loops; (c) the genome architecture and organization activating or suppressing genetic interactions between different parts of the genome. Most research so far has shed light on the puzzle pieces at these levels. This article, however, attempts an integrative approach to genome expression regulation incorporating these different layers. Under environmental stress or during cell development, differentiation towards specialized cell types, or to dysfunctional tumor, the cell nucleus seems to react as a whole through coordinated changes at all levels of control. This implies the need for a framework in which biological, chemical, and physical manifestations can serve as a basis for a coherent theory of gene self-organization. An international symposium held at the Biomedical Research and Study Center in Riga, Latvia, on 25 July 2022 addressed novel aspects of the abovementioned topic. The present article reviews the most recent results and conclusions of the state-of-the-art research in this multidisciplinary field of science, which were delivered and discussed by scholars at the Riga symposium.

Networks and Islands of Genome Nano-architecture and Their Potential Relevance for Radiation Biology : (A Hypothesis and Experimental Verification Hints).

The cell nucleus is a complex biological system in which simultaneous reactions and functions take place to keep the cell as an individualized, specialized system running well. The cell nucleus contains chromatin packed in various degrees of density and separated in volumes of chromosome territories and subchromosomal domains. Between the chromatin, however, there is enough "free" space for floating RNA, proteins, enzymes, ATPs, ions, water molecules, etc. which are trafficking by super- and supra-diffusion to the interaction points where they are required. It seems that this trafficking works somehow automatically and drives the system perfectly. After exposure to ionizing radiation causing DNA damage from single base damage up to chromatin double-strand breaks, the whole system "cell nucleus" responds, and repair processes are starting to recover the fully functional and intact system. In molecular biology, many individual epigenetic pathways of DNA damage response or repair of single and double-strand breaks are described. How these responses are embedded into the response of the system as a whole is often out of the focus of consideration. In this article, we want to follow the hypothesis of chromatin architecture's impact on epigenetic pathways and vice versa. Based on the assumption that chromatin acts like an "aperiodic solid state within a limited volume," functionally determined networks and local topologies ("islands") can be defined that drive the appropriate repair process at a given damage site. Experimental results of investigations of the chromatin nano-architecture and DNA repair clusters obtained by means of single-molecule localization microscopy offer hints and perspectives that may contribute to verifying the hypothesis.

Incorporation of Low Concentrations of Gold Nanoparticles: Complex Effects on Radiation Response and Fate of Cancer Cells.

(1) Background: In oncology research, a long-standing discussion exists about pros and cons of metal nanoparticle-enhanced radiotherapy and real mechanisms behind the tumor cell response to irradiation (IR) in presence of gold nanoparticles (GNPs). A better understanding of this response is, however, necessary to develop more efficient and safety nanoparticle (NP) types designed to disturb specific processes in tumor cells. (2) Aims and Methods: We combined 3D confocal microscopy and super-resolution single molecule localization microscopy (SMLM) to analyze, at the multiscale, the early and late effects of 10 nm-GNPs on DNA double strand break (DSB) induction and repair in tumor cells exposed to different doses of photonic low-LET (linear energy transfer) radiation. The results were correlated to different aspects of short and long-term cell viability. SkBr3 breast cancer cells (selected for the highest incidence of this cancer type among all cancers in women, and because most breast tumors are treated with IR) were incubated with low concentrations of GNPs and irradiated with 60Co γ-rays or 6 MV X-rays. In numerous post-irradiation (PI) times, ranging from 0.5 to 24 h PI, the cells were spatially (3D) fixed and labeled with specific antibodies against γH2AX, 53BP1 and H3K9me3. The extent of DSB induction, multi-parametric micro- and nano-morphology of γH2AX and 53BP1 repair foci, DSB repair kinetics, persistence of unrepaired DSBs, nanoscale clustering of γH2AX and nanoscale (hetero)chromatin re-organization were measured by means of the mentioned microscopy techniques in dependence of radiation dose and GNP concentration. (3) Results: The number of γH2AX/53BP1 signals increased after IR and an additional increase was observed in GNP-treated (GNP(+)) cells compared to untreated controls. However, this phenomenon reflected slight expansion of the G2-phase cell subpopulation in irradiated GNP(+) specimens instead of enhanced DNA damage induction by GNPs. This statement is further supported by some micro- and nano-morphological parameters of γH2AX/53BP1 foci, which slightly differed for cells irradiated in absence or presence of GNPs. At the nanoscale, Ripley's distance frequency analysis of SMLM signal coordinate matrices also revealed relaxation of heterochromatin (H3K9me3) clusters upon IR. These changes were more prominent in presence of GNPs. The slight expansion of radiosensitive G2 cells correlated with mostly insignificant but systematic decrease in post-irradiation survival of GNP(+) cells. Interestingly, low GNP concentrations accelerated DSB repair kinetics; however, the numbers of persistent γH2AX/53BP1 repair foci were slightly increased in GNP(+) cells. (4) Conclusions: Low concentrations of 10-nm GNPs enhanced the G2/M cell cycle arrest and the proportion of radiosensitive G2 cells, but not the extent of DNA damage induction. GNPs also accelerated DSB repair kinetics and slightly increased presence of unrepaired γH2AX/53BP1 foci at 24 h PI. GNP-mediated cell effects correlated with slight radiosensitization of GNP(+) specimens, significant only for the highest radiation dose tested (4 Gy).

Glucose Derivative Induced Vasculopathy in Children on Chronic Peritoneal Dialysis.

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Elucidation of the Clustered Nano-Architecture of Radiation-Induced DNA Damage Sites and Surrounding Chromatin in Cancer Cells: A Single Molecule Localization Microscopy Approach.

In cancer therapy, the application of (fractionated) harsh radiation treatment is state of the art for many types of tumors. However, ionizing radiation is a "double-edged sword"-it can kill the tumor but can also promote the selection of radioresistant tumor cell clones or even initiate carcinogenesis in the normal irradiated tissue. Individualized radiotherapy would reduce these risks and boost the treatment, but its development requires a deep understanding of DNA damage and repair processes and the corresponding control mechanisms. DNA double strand breaks (DSBs) and their repair play a critical role in the cellular response to radiation. In previous years, it has become apparent that, beyond genetic and epigenetic determinants, the structural aspects of damaged chromatin (i.e., not only of DSBs themselves but also of the whole damage-surrounding chromatin domains) form another layer of complex DSB regulation. In the present article, we summarize the application of super-resolution single molecule localization microscopy (SMLM) for investigations of these structural aspects with emphasis on the relationship between the nano-architecture of radiation-induced repair foci (IRIFs), represented here by γH2AX foci, and their chromatin environment. Using irradiated HeLa cell cultures as an example, we show repair-dependent rearrangements of damaged chromatin and analyze the architecture of γH2AX repair clusters according to topological similarities. Although HeLa cells are known to have highly aberrant genomes, the topological similarity of γH2AX was high, indicating a functional, presumptively genome type-independent relevance of structural aspects in DSB repair. Remarkably, nano-scaled chromatin rearrangements during repair depended both on the chromatin domain type and the treatment. Based on these results, we demonstrate how the nano-architecture and topology of IRIFs and chromatin can be determined, point to the methodological relevance of SMLM, and discuss the consequences of the observed phenomena for the DSB repair network regulation or, for instance, radiation treatment outcomes.

COMBinatorial Oligonucleotide FISH (COMBO-FISH) with Uniquely Binding Repetitive DNA Probes.

During the last decade, genome sequence databases of many species have been more and more completed so that it has become possible to further develop a recently established technique of FISH (Fluorescence In Situ Hybridization) called COMBO-FISH (COMBinatorial Oligo FISH). In contrast to standard FISH techniques, COMBO-FISH makes use of a bioinformatic search in sequence databases for probe design, so that it can be done for any species so far sequenced. In the original approach, oligonucleotide stretches of typical lengths of 15-30 nucleotides were selected in such a way that they only co-localize at the given genome target. Typical probe sets of about 20-40 stretches were used to label about 50-250 kb specifically. The probes of different lengths can be composed of purines and pyrimidines, but were often restricted to homo-purine or homo-pyrimidine probe sets because of the experimental advantage of using a protocol omitting denaturation of the target strand and triple strand binding of the probes. This allows for a better conservation of the 3D folding and arrangement of the genome. With an improved, rigorous genome sequence database analysis and sequence search according to statistical frequency and uniqueness, a novel family of probes repetitively binding to characteristic genome features like SINEs (Short Interspersed Nuclear Elements, e.g., ALU elements), LINEs (Long Interspersed Nuclear Elements, e.g., L1), or centromeres has been developed. These probes can be synthesized commercially as DNA or PNA probes with high purity and labeled by fluorescent dye molecules. Here, new protocols are described for purine-pyrimidine probes omitting heat treatment for denaturation of the target so that oligonucleotide labeling can also be combined with immune-staining by specific antibodies. If the dyes linked to the oligonucleotide stretches undergo reversible photo-bleaching (laser-induced slow blinking), the labeled cell nuclei can be further subjected to super-resolution localization microscopy for complex chromatin architecture research.

Challenges and Contradictions of Metal Nano-Particle Applications for Radio-Sensitivity Enhancement in Cancer Therapy.

From the very beginnings of radiotherapy, a crucial question persists with how to target the radiation effectiveness into the tumor while preserving surrounding tissues as undamaged as possible. One promising approach is to selectively pre-sensitize tumor cells by metallic nanoparticles. However, though the "physics" behind nanoparticle-mediated radio-interaction has been well elaborated, practical applications in medicine remain challenging and often disappointing because of limited knowledge on biological mechanisms leading to cell damage enhancement and eventually cell death. In the present study, we analyzed the influence of different nanoparticle materials (platinum (Pt), and gold (Au)), cancer cell types (HeLa, U87, and SKBr3), and doses (up to 4 Gy) of low-Linear Energy Transfer (LET) ionizing radiation (γ- and X-rays) on the extent, complexity and reparability of radiation-induced γH2AX + 53BP1 foci, the markers of double stand breaks (DSBs). Firstly, we sensitively compared the focus presence in nuclei during a long period of time post-irradiation (24 h) in spatially (three-dimensionally, 3D) fixed cells incubated and non-incubated with Pt nanoparticles by means of high-resolution immunofluorescence confocal microscopy. The data were compared with our preliminary results obtained for Au nanoparticles and recently published results for gadolinium (Gd) nanoparticles of approximately the same size (2⁻3 nm). Next, we introduced a novel super-resolution approach-single molecule localization microscopy (SMLM)-to study the internal structure of the repair foci. In these experiments, 10 nm Au nanoparticles were used that could be also visualized by SMLM. Altogether, the data show that different nanoparticles may or may not enhance radiation damage to DNA, so multi-parameter effects have to be considered to better interpret the radiosensitization. Based on these findings, we discussed on conclusions and contradictions related to the effectiveness and presumptive mechanisms of the cell radiosensitization by nanoparticles. We also demonstrate that SMLM offers new perspectives to study internal structures of repair foci with the goal to better evaluate potential differences in DNA damage patterns.

Recruitment of 53BP1 Proteins for DNA Repair and Persistence of Repair Clusters Differ for Cell Types as Detected by Single Molecule Localization Microscopy.

DNA double stranded breaks (DSBs) are the most serious type of lesions introduced into chromatin by ionizing radiation. During DSB repair, cells recruit different proteins to the damaged sites in a manner dependent on local chromatin structure, DSB location in the nucleus, and the repair pathway entered. 53BP1 is one of the important players participating in repair pathway decision of the cell. Although many molecular biology details have been investigated, the architecture of 53BP1 repair foci and its development during the post-irradiation time, especially the period of protein recruitment, remains to be elucidated. Super-resolution light microscopy is a powerful new tool to approach such studies in 3D-conserved cell nuclei. Recently, we demonstrated the applicability of single molecule localization microscopy (SMLM) as one of these highly resolving methods for analyses of dynamic repair protein distribution and repair focus internal nano-architecture in intact cell nuclei. In the present study, we focused our investigation on 53BP1 foci in differently radio-resistant cell types, moderately radio-resistant neonatal human dermal fibroblasts (NHDF) and highly radio-resistant U87 glioblastoma cells, exposed to high-LET 15N-ion radiation. At given time points up to 24 h post irradiation with doses of 1.3 Gy and 4.0 Gy, the coordinates and spatial distribution of fluorescently tagged 53BP1 molecules was quantitatively evaluated at the resolution of 10⁻20 nm. Clusters of these tags were determined as sub-units of repair foci according to SMLM parameters. The formation and relaxation of such clusters was studied. The higher dose generated sufficient numbers of DNA breaks to compare the post-irradiation dynamics of 53BP1 during DSB processing for the cell types studied. A perpendicular (90°) irradiation scheme was used with the 4.0 Gy dose to achieve better separation of a relatively high number of particle tracks typically crossing each nucleus. For analyses along ion-tracks, the dose was reduced to 1.3 Gy and applied in combination with a sharp angle irradiation (10° relative to the cell plane). The results reveal a higher ratio of 53BP1 proteins recruited into SMLM defined clusters in fibroblasts as compared to U87 cells. Moreover, the speed of foci and thus cluster formation and relaxation also differed for the cell types. In both NHDF and U87 cells, a certain number of the detected and functionally relevant clusters remained persistent even 24 h post irradiation; however, the number of these clusters again varied for the cell types. Altogether, our findings indicate that repair cluster formation as determined by SMLM and the relaxation (i.e., the remaining 53BP1 tags no longer fulfill the cluster definition) is cell type dependent and may be functionally explained and correlated to cell specific radio-sensitivity. The present study demonstrates that SMLM is a highly appropriate method for investigations of spatiotemporal protein organization in cell nuclei and how it influences the cell decision for a particular repair pathway at a given DSB site.

Dose enhancement effects of gold nanoparticles specifically targeting RNA in breast cancer cells.

Localization microscopy has shown to be capable of systematic investigations on the arrangement and counting of cellular uptake of gold nanoparticles (GNP) with nanometer resolution. In this article, we show that the application of specially modified RNA targeting gold nanoparticles ("SmartFlares") can result in ring like shaped GNP arrangements around the cell nucleus. Transmission electron microscopy revealed GNP accumulation in vicinity to the intracellular membrane structures including them of the endoplasmatic reticulum. A quantification of the radio therapeutic dose enhancement as a proof of principle was conducted with γH2AX foci analysis: The application of both-SmartFlares and unmodified GNPs-lead to a significant dose enhancement with a factor of up to 1.2 times the dose deposition compared to non-treated breast cancer cells. This enhancement effect was even more pronounced for SmartFlares. Furthermore, it was shown that a magnetic field of 1 Tesla simultaneously applied during irradiation has no detectable influence on neither the structure nor the dose enhancement dealt by gold nanoparticles.

Localization Microscopy Analyses of MRE11 Clusters in 3D-Conserved Cell Nuclei of Different Cell Lines.

In radiation biophysics, it is a subject of nowadays research to investigate DNA strand break repair in detail after damage induction by ionizing radiation. It is a subject of debate as to what makes up the cell's decision to use a certain repair pathway and how the repair machinery recruited in repair foci is spatially and temporarily organized. Single-molecule localization microscopy (SMLM) allows super-resolution analysis by precise localization of single fluorescent molecule tags, resulting in nuclear structure analysis with a spatial resolution in the 10 nm regime. Here, we used SMLM to study MRE11 foci. MRE11 is one of three proteins involved in the MRN-complex (MRE11-RAD50-NBS1 complex), a prominent DNA strand resection and broken end bridging component involved in homologous recombination repair (HRR) and alternative non-homologous end joining (a-NHEJ). We analyzed the spatial arrangements of antibody-labelled MRE11 proteins in the nuclei of a breast cancer and a skin fibroblast cell line along a time-course of repair (up to 48 h) after irradiation with a dose of 2 Gy. Different kinetics for cluster formation and relaxation were determined. Changes in the internal nano-scaled structure of the clusters were quantified and compared between the two cell types. The results indicate a cell type-dependent DNA damage response concerning MRE11 recruitment and cluster formation. The MRE11 data were compared to H2AX phosphorylation detected by γH2AX molecule distribution. These data suggested modulations of MRE11 signal frequencies that were not directly correlated to DNA damage induction. The application of SMLM in radiation biophysics offers new possibilities to investigate spatial foci organization after DNA damaging and during subsequent repair.

Challenges for Super-Resolution Localization Microscopy and Biomolecular Fluorescent Nano-Probing in Cancer Research.

Understanding molecular interactions and regulatory mechanisms in tumor initiation, progression, and treatment response are key requirements towards advanced cancer diagnosis and novel treatment procedures in personalized medicine. Beyond decoding the gene expression, malfunctioning and cancer-related epigenetic pathways, investigations of the spatial receptor arrangements in membranes and genome organization in cell nuclei, on the nano-scale, contribute to elucidating complex molecular mechanisms in cells and tissues. By these means, the correlation between cell function and spatial organization of molecules or molecular complexes can be studied, with respect to carcinogenesis, tumor sensitivity or tumor resistance to anticancer therapies, like radiation or antibody treatment. Here, we present several new applications for bio-molecular nano-probes and super-resolution, laser fluorescence localization microscopy and their potential in life sciences, especially in biomedical and cancer research. By means of a tool-box of fluorescent antibodies, green fluorescent protein (GFP) tagging, or specific oligonucleotides, we present tumor relevant re-arrangements of Erb-receptors in membranes, spatial organization of Smad specific ubiquitin protein ligase 2 (Smurf2) in the cytosol, tumor cell characteristic heterochromatin organization, and molecular re-arrangements induced by radiation or antibody treatment. The main purpose of this article is to demonstrate how nano-scaled distance measurements between bio-molecules, tagged by appropriate nano-probes, can be applied to elucidate structures and conformations of molecular complexes which are characteristic of tumorigenesis and treatment responses. These applications open new avenues towards a better interpretation of the spatial organization and treatment responses of functionally relevant molecules, at the single cell level, in normal and cancer cells, offering new potentials for individualized medicine.

Combining Low Temperature Fluorescence DNA-Hybridization, Immunostaining, and Super-Resolution Localization Microscopy for Nano-Structure Analysis of ALU Elements and Their Influence on Chromatin Structure.

Immunostaining and fluorescence in situ hybridization (FISH) are well established methods for specific labelling of chromatin in the cell nucleus. COMBO-FISH (combinatorial oligonucleotide fluorescence in situ hybridization) is a FISH method using computer designed oligonucleotide probes specifically co-localizing at given target sites. In combination with super resolution microscopy which achieves spatial resolution far beyond the Abbe Limit, it allows new insights into the nano-scaled structure and organization of the chromatin of the nucleus. To avoid nano-structural changes of the chromatin, the COMBO-FISH labelling protocol was optimized omitting heat treatment for denaturation of the target. As an example, this protocol was applied to ALU elements-dispersed short stretches of DNA which appear in different kinds in large numbers in primate genomes. These ALU elements seem to be involved in gene regulation, genomic diversity, disease induction, DNA repair, etc. By computer search, we developed a unique COMBO-FISH probe which specifically binds to ALU consensus elements and combined this DNA-DNA labelling procedure with heterochromatin immunostainings in formaldehyde-fixed cell specimens. By localization microscopy, the chromatin network-like arrangements of ALU oligonucleotide repeats and heterochromatin antibody labelling sites were simultaneously visualized and quantified. This novel approach which simultaneously combines COMBO-FISH and immunostaining was applied to chromatin analysis on the nanoscale after low-linear-energy-transfer (LET) radiation exposure at different doses. Dose-correlated curves were obtained from the amount of ALU representing signals, and the chromatin re-arrangements during DNA repair after irradiation were quantitatively studied on the nano-scale. Beyond applications in radiation research, the labelling strategy of immunostaining and COMBO-FISH with localization microscopy will also offer new potentials for analyses of subcellular elements in combination with other specific chromatin targets.

Smart Radiation Therapy Biomaterials.

Radiation therapy (RT) is a crucial component of cancer care, used in the treatment of over 50% of cancer patients. Patients undergoing image guided RT or brachytherapy routinely have inert RT biomaterials implanted into their tumors. The single function of these RT biomaterials is to ensure geometric accuracy during treatment. Recent studies have proposed that the inert biomaterials could be upgraded to "smart" RT biomaterials, designed to do more than 1 function. Such smart biomaterials include next-generation fiducial markers, brachytherapy spacers, and balloon applicators, designed to respond to stimuli and perform additional desirable functions like controlled delivery of therapy-enhancing payloads directly into the tumor subvolume while minimizing normal tissue toxicities. More broadly, smart RT biomaterials may include functionalized nanoparticles that can be activated to boost RT efficacy. This work reviews the rationale for smart RT biomaterials, the state of the art in this emerging cross-disciplinary research area, challenges and opportunities for further research and development, and a purview of potential clinical applications. Applications covered include using smart RT biomaterials for boosting cancer therapy with minimal side effects, combining RT with immunotherapy or chemotherapy, reducing treatment time or health care costs, and other incipient applications.

Cellular Uptake of Gold Nanoparticles and Their Behavior as Labels for Localization Microscopy.

Gold nanoparticles (GNPs) enhance the damaging absorbance effects of high-energy photons in radiation therapy by increasing the emission of Auger-photoelectrons in the nm-µm range. It has been shown that the incorporation of GNPs has a significant effect on radiosensitivity of cells and their dose-dependent clonogenic survival. One major characteristic of GNPs is also their diameter-dependent cellular uptake and retention. In this article, we show by means of an established embodiment of localization microscopy, spectral position determination microscopy (SPDM), that imaging with nanometer resolution and systematic counting of GNPs becomes feasible, because optical absorption and plasmon resonance effects result in optical blinking of GNPs at a size-dependent wavelength. To quantify cellular uptake and retention or release, SPDM with GNPs that have diameters of 10 and 25 nm was performed after 2 h and after 18 h. The uptake of the GNPs in HeLa cells was either achieved via incubation or transfection via DNA labeling. On average, the uptake by incubation after 2 h was approximately double for 10 nm GNPs as compared to 25 nm GNPs. In contrast, the uptake of 25 nm GNPs by transfection was approximately four times higher after 2 h. The spectral characteristics of the fluorescence of the GNPs seem to be environment-dependent. In contrast to fluorescent dyes that show blinking characteristics due to reversible photobleaching, the blinking of GNPs seems to be stable for long periods of time, and this facilitates their use as an appropriate dye analog for SPDM imaging.

Determining Omics spatiotemporal dimensions using exciting new nanoscopy techniques to assess complex cell responses to DNA damage: part A--radiomics.

Recent ground-breaking developments in Omics have generated new hope for overcoming the complexity and variability of biological systems while simultaneously shedding more light on fundamental radiobiological questions that have remained unanswered for decades. In the era of Omics, our knowledge of how genes and proteins interact in the frame of complex networks to preserve genome integrity has been rapidly expanding. Nevertheless, these functional networks must be observed with strong correspondence to the cell nucleus, which is the main target of ionizing radiation. Nuclear architecture and nuclear processes, including DNA damage responses, are precisely organized in space and time. Information regarding these intricate processes cannot be achieved using high-throughput Omics approaches alone, but requires sophisticated structural probing and imaging. Based on the results obtained from studying the relationship between higher-order chromatin structure, DNA double-strand break induction and repair, and the formation of chromosomal translocations, we show the development of Omics solutions especially for radiation research (radiomics) (discussed in this article) and how confocal microscopy as well as novel approaches of molecular localization nanoscopy fill the gaps to successfully place the Omics data in the context of space and time (discussed in our other article in this issue, "Determining Omics Spatiotemporal Dimensions Using Exciting New Nanoscopy Techniques to Assess Complex Cell Responses to DNA Damage: Part B--Structuromics"). Finally, we introduce a novel method of specific chromatin nanotargeting and speculate future perspectives, which may combine nanoprobing and structural nanoscopy to observe structure-function correlations in living cells in real time. Thus, the Omics networks obtained from function analyses may be enriched by real-time visualization of Structuromics.

Spatial distribution and structural arrangement of a murine cytomegalovirus glycoprotein detected by SPDM localization microscopy.

Novel approaches of localization microscopy have opened new insights into the molecular nano-cosmos of cells. We applied a special embodiment called spectral position determination microscopy (SPDM) that has the advantage to run with standard fluorescent dyes or proteins under standard preparation conditions. Pointillist images with a resolution in the order of 10 nm can be obtained by SPDM. Therefore, vector pEYFP-m164, encoding the murine cytomegalovirus glycoprotein gp36.5/m164 fused to enhanced yellow fluorescent protein, was transiently transfected into COS-7 cells. This protein shows exceptional intracellular trafficking dynamics, moving within the endoplasmic reticulum (ER) and outer nuclear membrane. The molecular positions of gp36.5/m164 were visualized and determined by SPDM imaging. From the position point patterns of the protein molecules, their arrangements were quantified by next neighbour distance analyses. Three different structural arrangements were discriminated: (a) a linear distribution along the membrane, (b) a highly structured distribution in the ER, and (c) a homogenous distribution in the cellular cytoplasm. The results indicate that the analysis of next neighbour distances on the nano-scale allows the identification and discrimination of different structural arrangements of molecules within their natural cellular environment.

Spatial allelic imbalance of BCL2 genes and chromosome 18 territories in nonneoplastic and neoplastic cervical squamous epithelium.

Several studies suggest a correlation between genome architecture and gene function. To elucidate mechanisms of gene positioning during cell differentiation and malignant transformation we investigated the nuclear positions of the BCL2 alleles and chromosome 18 territories in different layers of nonneoplastic cervical squamous epithelium and cervical squamous carcinomas in relation to gene expression. Fluorescence in situ hybridization and three-dimensional (3D) image analysis using tissue sections revealed that one BCL2 allele was located more peripherally than the other one in nuclei of the basal layer of nonneoplastic epithelium. During terminal cell differentiation the outer BCL2 allele showed a shift towards the nuclear center. In BCL2-expressing carcinomas the inner BCL2 allele was located more peripherally compared with the basal layer of nonneoplastic epithelium. Our results suggest a functional relevance of unequal allelic BCL2 gene positioning and support the hypothesis that transcriptional BCL2 activation is associated with BCL2 relocation towards the nuclear periphery.

Nano-sizing of specific gene domains in intact human cell nuclei by spatially modulated illumination light microscopy.

Although light microscopy and three-dimensional image analysis have made considerable progress during the last decade, it is still challenging to analyze the genome nano-architecture of specific gene domains in three-dimensional cell nuclei by fluorescence microscopy. Here, we present for the first time chromatin compaction measurements in human lymphocyte cell nuclei for three different, specific gene domains using a novel light microscopic approach called Spatially Modulated Illumination microscopy. Gene domains for p53, p58, and c-myc were labeled by fluorescence in situ hybridization and the sizes of the fluorescence in situ hybridization "spots" were measured. The mean diameters of the gene domains were determined to 103 nm (c-myc), 119 nm (p53), and 123 nm (p58) and did not correlate to the genomic, labeled sequence length. Assuming a spherical domain shape, these values would correspond to volumes of 5.7 x 10(-4) microm(3) (c-myc), 8.9 x 10(-4) microm(3) (p53), and 9.7 x 10(-4) microm(3) (p58). These volumes are approximately 2 orders of magnitude smaller than the diffraction limited illumination or observation volume, respectively, in a confocal laser scanning microscope using a high numerical aperture objective lens. By comparison of the labeled sequence length to the domain size, compaction ratios were estimated to 1:129 (p53), 1:235 (p58), and 1:396 (c-myc). The measurements demonstrate the advantage of the SMI technique for the analysis of gene domain nano-architecture in cell nuclei. The data indicate that chromatin compaction is subjected to a large variability which may be due to different states of genetic activity or reflect the cell cycle state.

COMBO-FISH: specific labeling of nondenatured chromatin targets by computer-selected DNA oligonucleotide probe combinations.

Here we present the principle of fluorescence in situ hybridization (FISH) with combinatorial oligonucleotide (COMBO) probes as a new approach for the specific labeling of genomic sites. COMBO-FISH takes advantage of homopurine/homopyrimidine oligonucleotides that form triple helices with intact duplex genomic DNA, without the need for prior denaturation of the target sequence that is usually applied for probe binding in standard FISH protocols. An analysis of human genome databases has shown that homopurine/homopyrimidine sequences longer than 14 bp are nearly homogeneously distributed over the genome, and they represent from 1% to 2% of the entire genome. Because the observation volume in a confocal laser-scanning microscope equipped with a high numerical aperture lens typically corresponds to an approximate 250-kb chromatin domain in a normal mammalian cell nucleus, this volume should contain 150-200 homopurine/homopyrimidine stretches. Using DNA database information, one can configure a set of distinct, uniformly labeled oligonucleotide probes from these stretches that is expected to exclusively co-localize within a 250-kb chromatin domain. Due to the diffraction-limited resolution of a microscope, the fluorescence signals of the configured oligonucleotide probe set merge into a typical, nearly homogenous FISH spot. Using a set of 32 homopyrimidine probes, we performed experiments in the Abelson murine leukemia region of human chromosome 9 as some of the very first proofs-of-principle of COMBO-FISH. Although the experimental protocol currently contains several steps that are incompatible with living cell conditions, the theoretical approach may be the first methodological advance toward the long-term but still elusive goal of carrying out specific FISH in high-resolution fluorescence microscopy of vital cells.